Objectives: 1.Establish neurogenesis models using Induced Pluripotent Stem cells (iPSC) and Induced Neural Stem Cells (iNSC) derived from patients for identifying novel genetic, epigenetic and other molecular targets for novel diagnosis and therapeutic interventions. -Screening assays for neurogenesis and neurotoxicity in human mixed T cells and neural cells co-cultures. -Identify and study the mechanisms of inflammation on neurogenesis and neurotoxicity. 2. Facilitate the utilization of our established iNSC models in basic and preclinical studies of neurological disorders by TNC, NIH and extramural investigators. Specific aim 1:Establish in vitro neurogenesis and development models using neural cultures derived from human adult peripheral CD34+ cells. We optimized our protocol and we can now derive both neural stem cells (iNS) and iPSC cells simultaneously from 10 ml of blood. We also studied the effect of certain small molecules and found that combination of retinoic receptor inhibitor G85 and small molecule UM171 will significantly inhibit CD34 differentiation and increase the cell number. Adoption of these small molecules will help us to better preserve CD34 cells in culture and provide more CD34 cells for T cells generation if needed. We also published another video protocol of culturing iNS from human CD34 cells on the Journal of Visualized Experiments (JoVE). Specific aim 2: Study the effect of inflammation on neural cells. We are using long term fluorescent cell tracer Vybrant CFDA SE to label T cells and cell membrane staining CM-Dil to label neural cells and created a dual fluorescence culture system to allow us to monitor T cell-induced neurotoxicity in a real-time manner. Using mixed T cell-neural cells cultures, we have found that T cells-derived from one undiagnosed patient blood showed neurotoxicity compared to control T cells from healthy donor. We are applying this in vitro neuroinflammatory model to the other T cell samples we collected from patients with undiagnosed neurological symptoms. This will help us to delineate whether the malfunction in T cells or abnormality in neuronal cells caused the symptoms. Specific aim 3: Study the effect of HERV K on pluripotent stem cell development. We continued the study of the effect of HERV-K Env on the pluripotent stem cells and neural differentiation. We confirmed that inhibition using siRNA or antibody against Env resulted in stem cell morphological changes and colony formation decrease. We further found that inhibition of HERV-K Env resulted in an enhanced neural induction process, likely due to its effect on the modulating of cell-cell adhesion through CD98HC. These finding suggested an important role HERV-K may have played in stem cell and human neurological system development. A paper is in prepared for publication based on these findings. Specific aim 4: Study the epigenetic mechanisms of neurodegenerative disorders. We are using induced neural stem cells generated from primary sclerosis (PLS) patients to study the possible effect of epigenetic changes in the pathogenesis of the disease. We found that general genomic DNA methylation changes during the neural stem cell induction and neuronal differentiation. The DNA methylation levels may be directly related to neuronal differentiation and maturation. Furthermore, neural stem cells derived from PLS samples showed slower neural differentiation and have lower DNA methylation level compared to normal controls, indicating epigenetic changes may play an important role in the pathogenesis of this disorder. We also collaborated with Dr. Roche to support the study on the possible role of neuroligin-4 in Autism which resulted in a recent publication in PNAS.